Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies

Nagy, Magdolna; Mastenbroek, Tom G.; Mattheij, Nadine J.A.; Witt, Susanne; Clemetson, Kenneth J.; Kirschner, Janbernd; Schulz, Ansgar; Vraetz, Thomas; Speckmann, Carsten; Braun, A.; Cosemans, Judith M.E.M.; Zieger, Barbara; Heemskerk, Johan W. M.

doi:10.3324/haematol.2017.176974

Cited by 37 publications

(55 citation statements)

References 33 publications

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“…The technique uses microspot-coated flow chambers for multiparameter measurement of thrombus formation during whole-blood perfusion at defined wall-shear rates. 7,9 In this study, we further standardized this method for inter-subject analysis of platelet function, using blood samples from healthy subjects, with results expected to represent the normal range observed in a population. Procedures included (see also the Online Supplement and de Witt et al .…”

Section: Resultsmentioning

confidence: 99%

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High-throughput elucidation of thrombus formation reveals sources of platelet function variability

et al. 2018

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In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: i.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced α IIb β 3 activation and secretion. Common sequence variation of GP6 and FCER1G , associated with GPVI-induced α IIb β 3 activation and secretion, affected parameters of GPVI-and CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.

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Section: Resultsmentioning

confidence: 99%

“…5,6 Recently, we showed that by using multiple, microspotted surfaces this method can be extended to multiparameter measurements of thrombus formation, 7 thus elucidating platelet dysfunction in patients with a range of bleeding diatheses. 7–9…”

Section: Introductionmentioning

confidence: 99%

High-throughput elucidation of thrombus formation reveals sources of platelet function variability

et al. 2018

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“…Previous studies using human blood showed an effect of low platelet counts on adhesion and activation in this assay, particularly on collagencoated surfaces. 17 Therefore, the observed reductions are due, at least in part, to thrombocytopenia in the loss-of-function and KO models.…”

Section: Increased Bleeding Is Caused By Aberrant Platelet Function Imentioning

confidence: 98%

Uncoupling ITIM receptor G6b-B from tyrosine phosphatases Shp1 and Shp2 disrupts murine platelet homeostasis

Geer

Geffen

Gopalasingam

et al. 2018

Blood

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The immunoreceptor tyrosine-based inhibitory motif (ITIM)-containing receptor G6b-B has emerged as a key regulator of platelet homeostasis. However, it remains unclear how it mediates its effects. Tyrosine phosphorylation of ITIM and immunoreceptor tyrosine-based switch motif (ITSM) within the cytoplasmic tail of G6b-B provides a docking site for Src homology 2 domain-containing protein-tyrosine phosphatases Shp1 and Shp2, which are also critical regulators of platelet production and function. In this study, we investigate the physiological consequences of uncoupling G6b-B from Shp1 and Shp2. To address this, we generated a transgenic mouse model expressing a mutant form of G6b-B in which tyrosine residues 212 and 238 within ITIM and ITSM were mutated to phenylalanine. Mice homozygous for the mutation () were macrothrombocytopenic, as a result of the reduction in platelet production, and had large clusters of megakaryocytes and myelofibrosis at sites of hematopoiesis, similar to those observed in -deficient mice and patients. Platelets from mice were hyporesponsive to collagen, as a result of the significant reduction in the expression of the immunoreceptor tyrosine-based activation motif (ITAM)-containing collagen receptor complex GPVI-FcR γ-chain, as well as thrombin, which could be partially rescued by costimulating the platelets with adenosine diphosphate. In contrast, platelets from ,, and megakaryocyte-specific mice were hyperresponsive to antibody-mediated cross-linking of the hemi-ITAM-containing podoplanin receptor CLEC-2, suggesting that G6b-B inhibits CLEC-2-mediated platelet activation through Shp2. Findings from this study demonstrate that G6b-B must engage with Shp1 and Shp2 to mediate its regulatory effects on platelet homeostasis.

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“…Numerical data of interventions are presented as means with SEM. Data from individual patients were compared, as described before 44 , to normal ranges established for a cohort of control subjects (set as means ± SD). Blood samples from patients and controls were analysed in parallel over the same period.…”

Section: Standardized Image Analysis and Delineation Of Thrombus Outcmentioning

confidence: 99%

Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation

Brouns

Geffen

Campello

et al. 2020

Sci Rep

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In haemostasis and thrombosis, platelet, coagulation and anticoagulation pathways act together to produce fibrin-containing thrombi. We developed a microspot-based technique, in which we assessed platelet adhesion, platelet activation, thrombus structure and fibrin clot formation in real time using flowing whole blood. Microspots were made from distinct platelet-adhesive surfaces in the absence or presence of tissue factor, thrombomodulin or activated protein C. Kinetics of platelet activation, thrombus structure and fibrin formation were assessed by fluorescence microscopy. This work revealed: (1) a priming role of platelet adhesion in thrombus contraction and subsequent fibrin formation; (2) a surface-independent role of tissue factor, independent of the shear rate; (3) a mechanism of tissue factor-enhanced activation of the intrinsic coagulation pathway; (4) a local, suppressive role of the anticoagulant thrombomodulin/protein C pathway under flow. Multiparameter analysis using blood samples from patients with (anti)coagulation disorders indicated characteristic defects in thrombus formation, in cases of factor V, XI or XII deficiency; and in contrast, thrombogenic effects in patients with factor V-Leiden. Taken together, this integrative phenotyping approach of platelet-fibrin thrombus formation has revealed interaction mechanisms of platelet-primed key haemostatic pathways with alterations in patients with (anti)coagulation defects. It can help as an important functional add-on whole-blood phenotyping. In the processes of haemostasis and thrombosis, platelet and coagulation pathways are tightly linked, with platelets both responding to thrombin and fibrin and, conversely, providing a phosphatidylserine-exposing surface on which high levels of thrombin and fibrin are formed 1-4. Thrombotic and bleeding disorders are mostly related to a dysregulation of one of these pathways, for instance when linked to platelet or coagulation factor mutations, or to the (combined) use of antiplatelet or anticoagulant medication. In the last decade, it has become clear that the accumulation of a platelet-and fibrin-containing plug or thrombus is instrumental to the onset of haemostatic as well as thrombotic events 5-7. An important modifying factor in thrombus development is provided by the local blood flow 8,9. This implies that, for adequate and comprehensive monitoring of the thrombotic process, measurements are to be performed under conditions of flow, preferentially recording both platelet and coagulation activation at the same time.

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Variable impairment of platelet functions in patients with severe, genetically linked immune deficiencies

Cited by 37 publications

References 33 publications

High-throughput elucidation of thrombus formation reveals sources of platelet function variability

High-throughput elucidation of thrombus formation reveals sources of platelet function variability

Uncoupling ITIM receptor G6b-B from tyrosine phosphatases Shp1 and Shp2 disrupts murine platelet homeostasis

Platelet-primed interactions of coagulation and anticoagulation pathways in flow-dependent thrombus formation

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